The discovery of antimicrobial peptides has unveiled a previously unrecognized component of animal host defense. We and others have discovered that mammalian mucosal epithelial cells express defensin peptides, providing them with the capacity to participate in mucosal host defense. In the human small intestine, defensins HD5 and HD6 are highly expressed in secretory epithelial cells called Paneth cells. Investigations by our group, and those of others, provide strong support for the hypothesis that Paneth cell defensin peptides provide a robust, critical defensive function in the intestine. Our studies have discovered also that HD5 is stored in Paneth cells as a modified propeptide. We identified that Paneth cell trypsin is the protease that processes proHD5 to the active antimicrobial present in the lumen. These data suggest that the proteolytic activity of Paneth call trypsin might be central to the regulation of enteric host defense. It is our hypothesis that 1) HD5 (and possibly HD6) is stored in Paneth cells as a glycosylated propeptide, providing a mechanism for both latent antibiotic activity and decreased toxicity to host cells, 2) HD6 has partially overlapping (or synergistic) antimicrobial functions with HD5 in the small intestine, and 3) once activated, Paneth cell trypsin activity unmasks latent antimicrobials and activates protease-activated receptor-2 (PAR-2) on enterocytes. In three specific aims, we will determine the posttranslational structural modifications of the tissue form of human proHD-5 (aim 1); establish the structure of tissue and luminal forms of HD-6 and determine their in vitro antimicrobial activity (aim 2); characterize Paneth cell trypsin isoforms, identify their key activities including processing of proHD-5 and -6, activation of other latent antimicrobials and activation of PAR-2, and identify the mechanism of Paneth cell protrypsin activation (aim 3). The proposed investigations may provide insights to novel therapeutic targets and approaches to combat infectious and inflammatory disease of the intestine.